Laser diffraction is a well-known technique for measuring sizes of particles in a dry powder state or suspended in a liquid suspension. A laser light is directed through the powder or suspension containing the particles to be measured. The particles cause the light to be diffracted, with the degree or angle of diffraction being dependent on the size of the particles in the suspension. By measuring the diffraction of the laser, it is thereby possible to calculate the size of the particles in the sample. The technique is described, for example, in the article “Particle Size analysis by laser diffraction: ISO 1332, Standard Operating Procedures, and Mie theory” by Rod Jones, published in American Laboratory, January 2003, pages 44-47.
Accurately measuring particle size, median particle size and particle size distribution is important, particularly in the pharmaceutical industry, where particle size is a critical quality attribute in determining how active drugs can be formulated, how stable the active drugs are and how bio-available the active drugs are. In certain applications, there is a requirement for the drug particles to be engineered to a median particle size target varying by no more than ±0.2 micron and even ±0.1 micron. This is an exquisite level of precision. In the pharmaceutical industry, precise and reproducible particle size measurements are necessary for manufacturing process development and validation, quality control purposes and generic product development.
There is therefore a need to be able to manufacture these particles to a very high level of precision, but this is only warranted if the analytical method is sufficiently precise to measure the particle size to the appropriate level of precision.
Unfortunately, unexplained variations in the precision of measurements have been observed since the method of laser diffraction for particle size analysis came into widespread use. These unexplained variations in the precision of the measurements are an obstacle to obtaining the benefits mentioned above and, more importantly, the variations prevent analytical laboratories from independently verifying and matching results obtained in another facility. Several theories have been advanced for these variations, such as algorithm artefacts in the interpretation of the diffraction of the laser beams, the chaotic nature of the flight of particles, the presence of bubble peaks, thermal artefacts, dry dispersion artefacts, optical model artefacts or the angle of incidence of the laser beam on the surface of the particles. Such problems are abundantly described in the literature (1, 2, 3, 4, 5) and solutions have been advanced. None of the solutions however have solved the precision issue of particle size measurement.
The research supporting the present application is oriented towards improving the de-agglomeration of tested particles.
It is known that the particles have a propensity to agglomerate or stick together. This is particularly true in the case of those particles which have been subjected to a size-reduction process, such as milling, jet mill micronization or wet polishing. The particles subjected to this size-reduction process demonstrate a very high level of cohesion, that is the particles stick together and form agglomerates of the particles. For this reason, it is preferred to suspend the particles in an appropriate suspension medium, which will promote the de-agglomeration of the particle clusters. In the absence of this suspension step, the particle size measurement performed by the laser diffraction equipment may result in inaccurate large measurement readings because the laser diffraction method cannot distinguish between large individual particles and smaller particles which have stuck together to form the larger agglomerate of the same size.
To de-agglomerate the suspended particles and to increase the precision of the particle size measurement, it is known to place the suspension in an ultrasonic bath, prior to the particle size measurement taking place. The ultra-sound bath contains an ultra-sound probe which excites the particles in the suspension and causes the particles to de-agglomerate from each other and hence causes the particles to separate from each other. This process is disclosed, for example, in European Patent No. EP 1 879 688 B1 (Orion), or International Patent Application No. WO 2008/016691 A2 (Covaris, Inc.).
During this de-agglomeration process, it is important that the ultra-sound probe is set to the correct voltage setting. If the voltage setting is set too low, then either the particles will not completely separate from each other or the process will take too long. If the voltage setting is set too high, the agitation of the particles can cause the individual particles to break down to a size smaller than that resulting from the manufacturing process. It has been found that in all these cases, this imperfect de-agglomeration will lead to an incorrect size measurement of the particles.
In the current art, the powders of interest intended for the particle size measurement need to undergo a sample preparation step to ensure that the measurement is likely to be more precise when the particles are suspended in a liquid medium, as opposed to the dry state. In a liquid suspension, the particle agglomerates need to be thoroughly dispersed, so that the laser beam will be diffracted by discrete particles and not by agglomerates of the particles. It will be appreciated that diffraction of the laser beam from the agglomerates of the particles will obviously result in incorrect size measurements.
For this purpose, samples of the powders of interest are usually placed in an appropriate anti-solvent in which the powders of interest may become suspended and a dispersant may be added to promote the de-agglomeration of the powders of interest. The suspension is placed in an ultra-sound bath comprising an ultra-sound probe connected to a power supply and a voltage controller, and the sample is sonicated for a set period, resulting in agglomerate dispersion of the powders.
In the laser diffraction apparatus that are commercially available, such as models manufactured by Malvern Instruments (Malvern, UK), the ultra-sound bath is provided within the apparatus itself, but the sample could equally be prepared in an ultra-sound bath independent of but conveniently adjacent to the apparatus. In such equipment, the voltage that is applied to the ultra-sound probe is given by an analogue representation of a power scale ranging from 0% to 100% of maximum power, in the software application of the equipment manufacturer. An analyst slides the power command control on the computer screen to vary the voltage of the ultra-sound probe. In equipment where the ultra-sound bath is not part of the laser diffraction equipment, the power can be set in many different ways. In certain models, ultra-sound baths function at a constant voltage and the variable to effect different sonication levels is simply the duration of the sonication. In other equipment, the power may be varied using a percent control of maximum power, or by setting a power level in watts or volts. In other words, the methods of setting ultra-sound power for the ultra-sound probe vary according to the equipment used.
Once the sample has been sonicated according to a validated method, the sample is introduced into the laser diffraction equipment and the measurement is made, yielding information about particle size, particle size distribution, surface area, etc.
To accurately measure the particle size of a powder of interest, two distinct steps are necessary. First, the analytical method must be developed and validated and each analytical method is specific to the product itself. This method development is important as the analytical method allows, in theory, the measurement to be repeated by different analysts using different laser diffraction equipment in different laboratories and to yield a valid result—in principle, the same test data should be obtained for the same batch of product.
In method development in the current art, the sonication step and its correct method development are important. To obtain the correct setting to analyze the particle size of a given compound, it is necessary to undergo a calibration procedure to obtain a correct power setting for the ultra-sound probe and to determine the correct sonication time for the suspension. It should be noted that this calibration will vary from product to product, as the products differ in terms of cohesiveness and in the way in which the products respond to sonication. The power for the head in the ultra-sound probe is set by a power controller command which is scaled from 0% to 100%—100% being maximum power. The power controller command is therefore set up so that the user selects the power level based on a percentage of the maximum rated power for the ultra-sound probe. For the method development and validation procedure, a power setting is selected on the power controller command and a sample suspension containing particles of a known size is exposed to the sonication or ultra-sound agitation. Size measurements by laser diffraction are taken and recorded at regular intervals until either complete de-agglomeration of the particles is confirmed, maximum time is reached or breakdown of the particles themselves is detected (measured particle size is less than the particle size of the sample, if the particle size is known).
The process is then completed at different power settings of the ultra-sound probe, and the results are analyzed by laser diffraction in order to determine the optimum power setting and exposure time for the particle type of the sample. The power setting is recorded as the percentage setting which should be set on the power controller command. This power setting is then recorded as part of the method development procedure and repeated using different batches of the same product, to obtain robust data upon which the applicable settings to use in the future when dealing with samples of the same product in the future, will be based. This is known as method development and validation and due to the time required to complete the procedure, it is normally only carried out when the new product is tested for the first time. Such validated settings are then usable, across the same type of ultra-sound baths and the laser diffraction particle size measurement machines.
After method development and validation of the prior art have been carried out, the second step of operation is routine analysis, in which the analyst consults the method development data for the product of interest, sets the ultra-sound probe and the ultra-sound head to the required power for sonication during the prescribed time, and then determines the particle size using the laser diffraction machine.
In theory, the determination of the particle size by laser diffraction, given a homogenous powder, should always yield the same result. In practice, it does not, with significant variations being observed when the same batch of powder is tested in different laser diffraction equipment. Indeed, it has been found that, on occasion, there is a significant deviation in the results obtained from different machines of the same model using the same settings on samples of the same batch of product. Tests carried out using two different machines do not always produce matching results, particularly when the need for precision is very high—as in the case of very small particle sizes such as are required for inhalation products. If the target median particle size is of 50 microns, then a variance of ±3 microns is not significant, but if the target particle size is 4 microns, then the same variance is extremely significant. Similarly, it has been found that the same machine can produce different results when testing different samples of the same batch of product at different times.
The problem is compounded by the fact that it occurs intermittently. On occasion, two different laser diffraction particle size analyzers have been found to produce different particle size test data for a same batch of powder. However, in other instances, notably at later test times—say months later—the same two analyzers may again agree and the test data are substantially the same from both analyzers.
It will be appreciated how the intermittent nature of this variability in the test data has had an important impact on analytical operations in the pharmaceutical industry. Laser diffraction is regarded as precise analytical method, and this is confirmed by widespread acceptance of the technology, not just for research and development purposes, but importantly for quality control. In this function, when a manufacturer supplies a product manufactured to a given particle specification and a customer needs to verify that the product does indeed meet its quality specification, disagreements over the test data have led to customers to reject batches of the product which apparently were failing to meet the required particle characteristics, at great cost to the manufacturer.
This issue has been known for more than 20 years, but previously no solution had been found. Sometimes users resorted to buying new ultra-sound probes, at great expense, but there was no scientific rationale for doing so. In other cases, analyst error was blamed after costly investigations. In extreme cases, particle analysis by laser diffraction would be replaced in the product specification by a less precise, but more reproducible method.
The present invention resides in the identification that the cause of this problem of non-reproducibility of results is due to differences and variability in the ultra-sound sonication arising from worn and/or damaged zones of the heads in the ultra-sound probes. It has been discovered that as the ultra-sound probes are used, the vibrating surfaces thereof become eroded and worn. The eroded and worn ultra-sound probes develop less energy and are therefore able to sonicate less, whereas defective or damaged ultra-sound probes may sonicate too much and break the particles. As a result, when the power for the ultra-sound probe is set on the power controller command using the percentage setting obtained during the method development and calibration process, the actual power delivered by the ultra-sound head has now been found, on occasion, to be different for different ones of the ultra-sound probes. In other words, the power that is set using the power controller command according to the method development and validation process does not always result in the same voltage being produced by the ultra-sound head. The inventors have found that this difference in delivered power for sonication explains the differences in particle size determinations that have been historically observed.
The need to control the voltage in an ultra-sound probe is known and there are several references in the prior art to this and to the control mechanisms employed in voltage adjustment. For instance, US patent application US20100191120A1 claims an ultra-sound system, wherein a sensor processor configured to generate a selection signal associated with an action when a detected level of at least one parameter associated with an object, is within a predetermined range of parameters including voltage. The ultra-sound system is controlled based on capacitance changes detected on the surface of the ultra-sound probe. However, the invention is directed at making user operation easier by reducing the number of manual commands and not at informing the user of the change in certain operating parameters, including voltage, to compensate for the loss of probe efficiency. Importantly, this prior art application does not contain any teaching that would enable the expert to identify voltage variations in sonication due to variable levels of ultra-sound probe corrosion as the root cause of imperfect dispersion of particle agglomerates and consequently poor reproducbility in particle size analysis by laser diffraction. Additionally, validation requirements in use the pharmaceutical industry would not have permitted automatically setting a sonication power level different from the power level determined in the method development and validation phase.
The difficulty in identifying the root cause of the variability lies in the fact that changes in the voltage produced by the aging probes occur over very long periods of time, so that unexplained differences in the particle size analysis test data have, to date, not been attributed to sonication and dispersion issues, and much less so to variability in voltage output from the ultra-sound probes.